PCR diagnostics.eu - HHV7-screen-monitor-FRT ME …...PRINCIPLE OF PCR DETECTION The principle of testing is based on the DNA extraction from test samples together with the exogenous

For Professional Use Only

AmpliSens HHV7-screen/monitor-FRT

PCR kit

Instruction Manual

AAmmpplliiSSeennss

Ecoli s.r.o., Studenohorska 12 841 03 Bratislava 47 Slovak Republic Tel.: +421 2 6478 9336 Fax: +421 2 6478 9040

Federal Budget Institute of Science “Central Research Institute for Epidemiology” 3A Novogireevskaya Street Moscow 111123 Russia

REF H-2431-1-1-CE / VER 02.10.17–04.04.18 / Page 2 of 18

TABLE OF CONTENTS

1. INTENDED USE............................................................................................................. 3

2. PRINCIPLE OF PCR DETECTION ................................................................................ 3

3. CONTENT ...................................................................................................................... 4

4. ADDITIONAL REQUIREMENTS .................................................................................... 5

5. GENERAL PRECAUTIONS ........................................................................................... 6

6. SAMPLING AND HANDLING ......................................................................................... 7

7. WORKING CONDITIONS .............................................................................................. 8

8. PROTOCOL ................................................................................................................... 8

9. DATA ANALYSIS ......................................................................................................... 11

10. TROUBLESHOOTING ............................................................................................... 13

11. TRANSPORTATION .................................................................................................. 14

12. STABILITY AND STORAGE ...................................................................................... 14

13. SPECIFICATIONS ..................................................................................................... 14

14. REFERENCES ........................................................................................................... 16

15. QUALITY CONTROL ................................................................................................. 17

16. KEY TO SYMBOLS USED ......................................................................................... 18


1. INTENDED USE

AmpliSens® HHV7-screen/monitor-FRT PCR kit is an in vitro nucleic acid amplification

test for quantitative detection of human herpes virus type 7 (HHV7) DNA in the biological

material (blood plasma, whole blood, saliva), taken from the persons suspected of herpes

virus infection, using real-time hybridization-fluorescence detection of amplified products.

The results of PCR analysis are taken into account in complex diagnostics of disease.

2. PRINCIPLE OF PCR DETECTION

The principle of testing is based on the DNA extraction from test samples together with the

exogenous internal control (Internal Control-FL (IC)) and simultaneous amplification of

DNA fragments of the detected microorganism and DNA of the exogenous and

endogenous internal control with hybridization-fluorescence detection. DNA extraction

from blood plasma, whole blood and saliva is carried out in the presence of the exogenous

internal control (Internal Control-FL (IC)) in order to control all PCR-analysis stages of

each individual sample and to identify possible reaction inhibition. When extracting from

whole blood the amplification of the human β-globin gene DNA fragment (endogenous

internal control) is carried out. The use of the endogenous internal control (IC Glob) makes

it possible not only to monitor test stages but also to assess the adequacy of sampling and

storage of material.

HHV7 detection by the polymerase chain reaction (PCR) is based on the amplification of

the pathogen genome specific region using specific HHV7 primers. In the real-time PCR,

the amplified product is detected with the use of fluorescent dyes. These dyes are linked to

oligonucleotide probes, which bind specifically to the amplified product during

thermocycling. The real-time monitoring of fluorescence intensities during the real-time

PCR allows the detection of accumulating product without re-opening the reaction tubes

after the PCR run.

The quantitative analysis of HHV7 DNA is based on the linear dependence between the

initial concentration of DNA target in a test sample and the cycle threshold (Ct) (the cycle

of beginning of fluorescence signal exponential growth). For the quantitative analysis

amplification of DNA from the test samples is carried out simultaneously with DNA-

calibrators (samples with the known concentration of the DNA target). Based on the

amplification results of DNA-calibrators a calibration line is plotted and it is used for the

estimation of concentration of the DNA target in the test samples.


The PCR kit contains the system for prevention of contamination by amplicons using the

enzyme uracil-DNA-glicosylase (UDG) and deoxyuridine triphosphate. The enzyme UDG

recognizes and catalyzes the destruction of the DNA containing deoxyuridine, but has no

effect on DNA containing deoxythymidine. Deoxyuridine is absent in the authentic DNA,

but is always present in amplicons, because deoxyuridine triphosphate is a part of dNTP

mixture in the reagents for the amplification. Due to the deoxyuridine containing

contaminating amplicons are sensitive to the destruction by UDG before the DNA-target

amplification. So the amplicons cannot be amplified.

The enzyme UDG is thermolabile. It is inactivated by heating at temperature above 50 °C.

Therefore, UDG does not destroy the target amplicons which are accumulated during

PCR.

At the amplification stage 3 reactions are carried out in one tube simultaneously:

amplification of HHV7 DNA as well as amplification of Internal Control-FL (IC) DNA and

human DNA fragment (IC Glob). The results of amplification of HHV7 DNA as well as

Internal Control-FL (IC) and IC Glob DNA are registered in 3 different fluorescence

channels.

Table 1

Channel for fluorophore

FAM JOE ROX

DNA-target human DNA (IC Glob) HHV7 DNA Internal Control-FL

(IC) DNA

Target gene β-globin gene

fragment DNA fragment of HHV7 MCP-gene

artificially synthesized sequence

3. CONTENT

AmpliSens® HHV7-screen/monitor-FRT PCR kit is produced in 1 form:

AmpliSens® HHV7-screen/monitor-FRT PCR kit variant FRT-100 FN,

REF H-2431-1-1-CE

AmpliSens® HHV7-screen/monitor-FRT PCR kit variant FRT-100 FN includes:

Reagent Description Volume, ml Quantity

PCR-mix-FL HHV7 clear liquid from colorless to light lilac colour

1.2 1 tube

PCR-buffer-H colorless clear liquid 0.6 1 tube

С1 HHV7 colorless clear liquid 0.2 1 tube

С2 HHV7 colorless clear liquid 0.2 1 tube

TE-buffer colorless clear liquid 0.2 1 tube

Internal Control-FL (IC)* colorless clear liquid 1.0 1 tube


Reagent Description Volume, ml Quantity

Negative Control (C–)** colorless clear liquid 1.2 2 tubes

Positive Control HHV7*** colorless clear liquid 0.1 1 tube

* add 10 µl of Internal Control-FL (IC) during the DNA extraction procedure directly to

the sample/lysis mixture (see RIBO-prep, REF K2-9-Et-100-CE protocol).

** must be used in the extraction procedure as Negative Control of Extraction.

*** must be used in the extraction procedure as Positive Control of Extraction.

AmpliSens® HHV7-screen/monitor-FRT PCR kit variant FRT-100 FN is intended for

110 reactions (including controls).

4. ADDITIONAL REQUIREMENTS

Vacuette® blood collection system.

Medical centrifuge with equipment.

Reagent for pretreatment of whole peripheral and umbilical blood.

Microcentrifuge for Eppendorf tubes (RCF max. 12,000 x g).

Vacuum aspirator with flask for removing supernatant.

DNA extraction kit.

Disposable powder-free gloves and a laboratory coat.

Pipettes (adjustable).

Sterile pipette tips with filters (up to 100, 200 and 1,000 µl).

Tube racks.

Vortex mixer.

PCR box.

Real-time instruments (for example, Rotor-Gene Q (QIAGEN GmbH, Germany);

CFX 96 (Bio-Rad Laboratories, Inc., USA)).

Disposable polypropylene tubes:

a) tightly closed 2.0-ml tubes for sampling.

b) tightly closed 1.5-ml tubes for pretreatment.

c) tightly closed 1.5-ml tubes for reaction mixture preparation.

d) thin-walled 0.2-ml PCR tubes with optical transparent domed or flat caps or strips of

eight 0.2-ml tubes with optical transparent caps if a plate-type instrument is used;

e) thin-walled 0.2-ml PCR tubes with flat caps or strips of four 0.1-ml Rotor-Gene PCR

tubes if a rotor-type instrument is used.

Refrigerator for 2–8 °C.


Deep-freezer at the temperature from minus 24 to minus 16 °C.

Reservoir for used tips.

5. GENERAL PRECAUTIONS

The user should always pay attention to the following:

Use sterile pipette tips with aerosol filters and use a new tip for every procedure.

Store all extracted positive material (specimens, controls and amplicons) away from all

other reagents and add it to the reaction mix in a distantly separated facility.

Thaw all components thoroughly at room temperature before starting an assay.

When thawed, mix the components and centrifuge briefly.

Use disposable protective gloves and laboratory cloths, and protect eyes while samples

and reagents handling. Thoroughly wash hands afterwards.

Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work

areas.

Do not use the PCR kit if the internal packaging was damaged or its appearance was

changed.

Do not use the PCR kit if the transportation and storage conditions according to the

Instruction Manual were not observed.

Do not use a kit after its expiration date.

Dispose of all specimens and unused reagents in accordance with local regulations.

Samples should be considered potentially infectious and handled in biological cabinet in

compliance with appropriate biosafety practices.

Clean and disinfect all samples or reagents spills using a disinfectant, such as 0.5 %

sodium hypochlorite or another suitable disinfectant.

Avoid samples and reagents contact with the skin, eyes, and mucous membranes. If

these solutions come into contact, rinse the injured area immediately with water and

seek medical advice immediately.

Safety Data Sheets (SDS) are available on request.

The PCR kit is intended for single use for PCR analysis of specified number of samples

(see the section “Content”).

The PCR kit is ready for use in accordance with the Instruction Manual. Use the PCR

kit strictly for intended purpose.

Use of this product should be limited to personnel trained in DNA amplification

techniques.

Workflow in the laboratory must be one-directional, beginning in the Extraction Area


and moving to the Amplification and Detection Area. Do not return samples, equipment

and reagents in the area where the previous step was performed.

Some components of this kit contain sodium azide as a preservative. Do not use metal tubing for reagent transfer.

6. SAMPLING AND HANDLING

AmpliSens® HHV7-screen/monitor-FRT PCR kit is intended for analysis of the DNA

extracted with DNA extraction kits from the biological material (blood plasma, whole blood,

saliva).

Sampling

6.1 Blood plasma. To obtain the plasma samples, blood should be taken after overnight

fasting or in 3 hour after eating by a disposable 0.8-1.1 mm diameter needle into the tube

with EDTA (special vacuum system Vacuette® (lavender caps – 6 % EDTA)). After blood

sampling the tube should be gently inverted several times for the thoroughly mixing with

the anticoagulant. During 6 hours after blood sampling plasma should be transferred into a

new tube. To do this the tubes with whole blood should be centrifuged at 800 g for 10 min

at room temperature. No less than 1 ml of obtained plasma is transferred by separate filter

tips into sterile dry 2.0-ml tubes.

The samples can be stored before the DNA extraction:

– at the temperature from 2 to 8 °С – for 2 days,

– at the temperature from minus 24 to minus 16 °С – for 1 year.

Only one freeze-thaw cycle is allowed.

6.2 Whole blood. Blood should be taken after overnight fasting or in 3 hour after eating

by a disposable 0.8-1.1 mm diameter needle into the tube with EDTA (special vacuum

system Vacuette® (lavender caps – 6 % EDTA)). After blood sampling the tube should be

gently inverted several times for the thoroughly mixing with the anticoagulant. (Otherwise,

blood will coagulate and DNA extraction will be impossible!)

The samples can be stored before the pretreatment:


6.3 Saliva should be obtained after rinsing the oral cavity 3 times with saline solution.

Take saliva in sterile dry 2.0 ml tubes in an amount not less than 0.5 ml. Cap the tube

tightly, preventing the gap and crumbling the inside of the cap, and mark it.

The samples can be stored before the DNA extraction:





Pretreatment

6.4 Pretreatment of blood plasma and saliva samples is not required.

6.7 Whole blood samples are to be prepared. Transfer 0.25 ml of whole blood to the

disposable 1.5-ml tube. Add 1.0 ml of Hemolytic REF 137-CE. Gently vortex the tubes

and leave them for 10 minutes at room temperature (from 18 to 25оС), stirring

occasionally. Centrifuge at 4,000 g (8,000 rpm) for 3 min. Remove the supernatant using

vacuum aspirator leaving 100 µl of the pellet. After washing the cell pellet should be white,

only a small pinkish bloom on the pellet is allowed (the remains of the destroyed

erythrocytes). The washing using Hemolytic REF 137-CE may be repeated if necessary.

The obtained leucocytes pellet must be immediately lysed (in case of extraction using

RIBO-prep add 300 µl of Solution for Lysis and then extract DNA in accordance with the

Instruction Manual enclosed to the RIBO-prep reagent kit without adding Solution for

Lysis once again).

The whole blood samples prepared using Solution for Lysis can be stored before the PCR:



Interfering substances and limitations of using test material samples

In order to control the DNA extraction efficiency and possible reaction inhibition the

Internal Control (Internal Control-FL (IC)) is used in the PCR kit. The Internal Control is

added in each biological sample at the extraction stage. The presence of internal control

signal after the amplification testifies the effectiveness of nucleic acid extraction and the

absence of PCR inhibitors.

The next samples are inapplicable for analysis:

– the whole blood samples, collected in the tubes with heparin as anticoagulant,

– the whole blood samples, containing blood clot or which has been exposed to freezing.

7. WORKING CONDITIONS

AmpliSens® HHV7-screen/monitor-FRT PCR kit should be used at 18–25 °C.

8. PROTOCOL

8.1. DNA extraction

It is recommended to use the following nucleic acid extraction kits:

RIBO-prep, REF K2-9-Et-100-CE.


If using the RIBO-prep kit extract the DNA according to the manufacturer’s protocol.

The volumes of reagents and samples when the DNA is extracted by the RIBO-prep reagent kit:

The DNA extraction for each sample is carried out in the presence of Internal Control-FL (IC).

Add 10 µl of Internal Control-FL (IC) to each tube.

The volume of the test sample is 100 µl.

Add 100 µl of Negative Control (C–) into the tube labeled C– (Negative Control of Extraction).

Add 10 µl of Positive Control HHV7 and 90 µl of Negative Control (C–) into the tube labeled PCE (Positive Control of Extraction).

The volume of elution is 50 µl.

8.3. Preparing PCR

8.3.1 Preparing tubes for PCR

The total reaction volume is 25 µl, the volume of the DNA sample is 10 µl.

The type of tubes depends on the PCR instrument used for analysis. Use disposable filter

tips for adding reagents, DNA and control samples into tubes.

1. Calculate the required quantity of each reagent for reaction mixture preparation. For one

reaction:

10 µl of PCR-mix-FL HHV7,

5 µl of PCR-buffer-H.

Prepare the reaction mixture for the total number of test and control samples plus several

extra reactions. Number of control samples see in item 7.

Prepare the reaction mixture just before use.

2. Thaw the tubes with PCR-mix-FL HHV7 and PCR-buffer-H. Thoroughly vortex the

tubes with PCR-mix-FL HHV7 and PCR-buffer-H and sediment the drops by vortex.

3. In a new tube prepare the reaction mixture. Mix the required quantities of PCR-mix-FL

HHV7 and PCR-buffer-H. Sediment the drops by vortex.

4. Take the required number of the tubes or strips taking into account the number of test

samples and control samples.

5. Transfer 15 µl of the prepared reaction mixture to each tube. Discard the unused

reaction mixture.

6. Add 10 µl of DNA samples extracted from test samples at the DNA extraction stage

using tips with filter.


7. Carry out the control reactions:

C1 Add 10 µl of С1 HHV7 to the tube labeled C1.

C2 Add 10 µl of С2 HHV7 to the tube labeled C2.

C– Add 10 µl of the sample extracted from the Negative Control (C–) reagent to the tube labeled C– (Negative control of Extraction).

PCE Add 10 µl of the sample extracted from the Positive Control HHV7 reagent to the tube labeled PCE (Positive control of Extraction).

It is also necessary to carry out Negative Control of Amplification (NCA) at suspicion on possible contamination

NCA Add 10 µl of TE-buffer to the tube with reaction mixture.

8.3.2. Amplification

1. Create a temperature profile on your instrument as follows:

Table 2

AmpliSens unified amplification program for rotor-1 and plate-type2 instruments

Step Temperature, °С Time Fluorescent signal detection Cycles

1 50 15 min – 1

2 95 15 min – 1

3 95 10 s –

45 60 20 s FAM, JOE, ROX

Any combination of the tests (including tests with reverse transcription and amplification) can be performed in one instrument simultaneously with the use of the unified amplification program. If several tests in “multiprime” format are carried out simultaneously, the detection is enabled in other used channels except for the specified ones. If in one instrument only the tests for the pathogen DNA detection are carried out simultaneously, the first step of reverse transcription (50 °С – 15 min) can be omitted for time saving.

2. Adjust the fluorescence channel sensitivity according to the Important Product

Information Bulletin and Guidelines [2].

3. Insert tubes into the reaction module of the device.

It is recommended to sediment drops from walls of tubes by short centrifugation (1–3 s) before placing them into the instrument. Insert empty tubes at the edges of reaction module in case of incomplete filling of plate-type instrument.

4. Run the amplification program with fluorescence detection.

5. Analyze results after the amplification program is completed.

1 For example, Rotor-Gene Q (QIAGEN, Germany).

2 For example, CFX 96 (Bio-Rad, USA).


9. DATA ANALYSIS

Analysis of results is performed by the software of the real-time PCR instrument used by

measuring fluorescence signal accumulation in three channels:

Table 3

Channel for the fluorophore FAM JOE ROX

Signal registration, indicating the amplification product accumulation

human DNA (IC Glob)

HHV7 DNA Internal Control-FL

(IC) DNA

Results are interpreted by the crossing (or not-crossing) the fluorescence curve with the

threshold line set at the specific level that corresponds to the presence (or absence) of a

Ct value of the DNA sample in the corresponding column of the results grid.

Based on the obtained Ct values and specified concentration values of DNA calibrators

(C1 and C2) a calibration line is plotted and the concentration values of HHV7 DNA,

human DNA (IC Glob) and Internal Control-FL (IC) DNA in copies/reaction are calculated.

HHV7 DNA quantity per 1 ml is calculated according to the formula:

number of HHV7 DNA copies per reaction x coefficient В = GE/ml

number of Internal Control-FL (IC) DNA copies per reaction

where;

Coefficient В is number of genome equivalents (GE) of IC in 1 ml of the test sample. The

coefficient takes into account the DNA loss during the extraction procedure.

The values of calibrators’ concentrations and coefficient В are specified in the Important Product Information Bulletin enclosed in the PCR kit and is specific for each lot. It cannot be used with PCR kits of different lots.

When DNA is extracted from whole blood samples, the obtained HHV7 DNA concentration

values can be normalized to the standard number of human cells (the number of HHV7 GE

per 105 of human cells). Normalized HHV7 DNA concentration values are calculated

according to the formula:

number of HHV7 DNA copies per reaction

number of IC Glob DNA copies per reaction

х 2*105 = number of HHV7 GE per

105 of human cells

Normalized concentration values reflect the number of GE of the pathogen relative to

human cells.

To express the relative concentration of HHV7 DNA in GE per the standard number of cells,

the conversion factors are used:

105 of cells = 2*105 of human genomes;

The values of calibrators’ concentrations are specified in the Important Product Information Bulletin enclosed to the PCR kit.


Table 4

Results Interpretation for the test samples

Result Interpretation

Invalid

The Ct value in the channel for the ROX fluorophore is absent or determined

greater than the boundary value. The PCR analysis (beginning with the DNA

extraction stage) should be repeated for this sample.

Invalid (for the whole blood

analysis only)

IC Glob concentration is less than 2,000 copies/reaction and the value of

calculated concentration is absent in the channel for the JOE fluorophore. The

PCR analysis (beginning with the DNA extraction stage) should be repeated for

this sample.

HHV7 DNA is not

detected

The Ct value for HHV7 DNA is absent and the Ct value determined in the

channel for the ROX fluorophore is less than the boundary value. The result is

HHV7 DNA is not detected.

less than 1,000 GE/ml

Detected HHV7 DNA concentration is less than the linear range of the PCR kit.

The result is less than 1,000 HHV7 GE/ml

X х 10y GE/ml

Calculated concentration value (GE/ml) is in the linear measurement range of

the PCR kit. The result is HHV7 DNA is detected at a concentration of X х

10y GE/ml

greater than 1x107 GE/ml

Detected HHV7 DNA concentration is greater than the linear measurement

range of the PCR kit. The result is greater than 1x107 HHV7 GE/ml. If the

accurate quantification is required, the extracted sample is to be diluted by TE-

buffer reagent (for example, 100-fold dilution) and the PCR-analysis is to be

repeated from the amplification stage. The result obtained after repeated

analysis should be multiplied by the coefficient of the sample dilution.

The result of the analysis is considered reliable only if the results obtained for the

controls of extraction and amplification are correct (see Table 5).

Table 5

Results for controls

Control Stage for control

Amplification results in the channel for fluorophore

FAM JOE ROX

PCE DNA extraction Ct value < boundary

value

Ct value < boundary value, concentration value is within the

range

Ct value < boundary value

C– DNA extraction Ct value is absent Ct value is absent Ct value < boundary

value

NCA PCR Ct value is absent Ct value is absent Ct value is absent

С1 PCR Ct value and calculated

concentration are determined

Ct value and calculated concentration are

determined


determined

С2 PCR Ct value and calculated

concentration are determined


determined


determined

Boundary Ct values and the range of Positive Control HHV7 concentration are specified in the Important Product Information Bulletin enclosed to the PCR kit.


10. TROUBLESHOOTING

Results of analysis are not taken into account in the following cases:

1. The Ct value determined for the Positive Control of Extraction (PCE) in the channels for

the FAM and/or JOE and/or ROX fluorophores is greater than the boundary Ct value or

absent. The PCR analysis (beginning with the DNA extraction stage) should be

repeated for all samples.

2. The calculated concentration of the Positive Control HHV7 does not fit in the range

specified in the bulletin. The PCR analysis (beginning with the DNA extraction stage)

should be repeated for all samples.

3. The Ct value is determined for the Negative Control of Extraction (C–) in the channels

for the FAM and/or JOE fluorophores. The contamination of laboratory with

amplification fragments or contamination of reagents, test samples is probable at any

stage of PCR analysis. Measures for detecting and elimination of contamination source

must be taken. The PCR analysis (beginning with the DNA extraction stage) should be

repeated for all samples in which specific DNA was detected.

4. The Ct value is determined for the Negative Control of amplification (NCA) in the

channels for the FAM and/or JOE and/or ROX fluorophores. The contamination of

laboratory with amplification fragments or contamination of reagents, test samples is

probable at any stage of PCR analysis. Measures for detecting and elimination of

contamination source must be taken. The amplification and detection should be

repeated for all samples in which specific DNA was detected.

5. The Ct values are absent for the DNA-calibrators C1 and C2 in either of the specified

channels for fluorophores. The amplification and detection should be repeated for all

the samples.

6. The correlation coefficient R2 is less than 0.98 when plotting the calibration curve.

Check the correctness of set concentrations of calibrators in accordance with the

bulletin. If the improper result has been obtained again the amplification and detection

for all the samples should be repeated.

7. The Ct value is determined for the test sample, whereas the area of typical exponential

growth of fluorescence is absent (the graphic looks like approximate straight line). It is

necessary to check the correctness of selected threshold line level or parameters of

base line calculation. If the result has been obtained with the correct level of threshold

line (base line), the amplification and detection should be repeated for this sample.

If you have any further questions or if you encounter problems, please contact our

Authorized representative in the European Community.


11. TRANSPORTATION

AmpliSens® HHV7-screen/monitor-FRT PCR kit should be transported at 2–8 °C for no

longer than 5 days. PCR kit can be transported at 2–25 °C for no longer than 3 days.

12. STABILITY AND STORAGE

All components of the AmpliSens® HHV7-screen/monitor-FRT PCR kit are to be stored at

2–8 °C when not in use (except for PCR-buffer-H and PCR-mix-FL HHV7). All components

of the AmpliSens® HHV7-screen/monitor-FRT PCR kit are stable until the expiry date

stated on the label. The shelf life of reagents before and after the first use is the same,

unless otherwise stated.

PCR-buffer-H and PCR-mix-FL HHV7 are to be stored at the temperature from minus 24 to minus 16 °C

PCR-mix-FL HHV7 is to be kept away from light

13. SPECIFICATIONS

13.1. Linear range and limit of detection

Table 6

Biological material

The volume of sample for extraction, µl

Nucleic acid extraction kit

PCR kit Limit of

detection, GE/ml

Linear measurement range, GE/ml

Blood plasma

100 RIBO-prep PCR kit variant FRT-100 FN

5x102 1x103 – 1x107 Whole blood

Saliva

The claimed features are achieved while respecting the rules specified in the section

“Sampling and Handling”.

13.2. Analytical specificity

The analytical specificity of AmpliSens® HHV7-screen/monitor-FRT PCR kit is ensured

by the selection of specific primers and probes as well as stringent reaction conditions.

The primers and probes have been checked for possible homologies to all sequences

published in gene banks by sequence comparison analysis.

The PCR kit detects the DNA fragments of claimed microorganisms. The specificity was

proved on the following strains of microorganisms: ATCC® 19606TM Acinetobacter

baumanii, ATCC® 29212TM Enterococcus faecalis, ATCC® 25922TM Escherichia coli,

ATCC® 33930TM Haemophilus influenzae, ATCC® 27736TM Klebsiella pneumoniae, ATCC®

25401TM Listeria grayi (murrayi), ATCC® 7644TM Listeria monocytogenes, ATCC® 33090TM

Listeria innocua, ATCC® 15442TM Pseudomonas aeruginosa, ATCC® 6538PTM


Staphylococcus aureus, ATCC® 43300TM Staphylococcus aureus (MRSA), ATCC®

12228TM Staphylococcus epidermidis, ATCC® 29970TM Staphylococcus haemolyticus,

ATCC® 49907TM Staphylococcus saprophyticus, ATCC® 12386TM Streptococcus

agalactiae, ATCC® 19615TM Streptococcus pyogenes, ATCC® 25240TM Moraxella

catarrhalis, HSV I (Herpes simplex virus type 1), HSV II (Herpes simplex virus type 2),

CMV (human cytomegalovirus), EBV (Epstein-Barr virus), HHV6 (human herpesvirus

type 6), HHV8 (human herpesvirus type 8), VZV (varicella zoster virus), JCV (John

Cunningham virus), Parvovirus B19, Toxoplasma gondii, Candida albicans, and also

human genomic DNA.

The nonspecific responses were not observed while testing the DNA samples of the above

mentioned microorganisms, as well as human DNA.

The clinical specificity of AmpliSens® HHV7-screen/monitor-FRT PCR kit was confirmed

in laboratory clinical trials.

13.3. Reproducibility, repeatability and trueness

Repeatability and reproducibility were determined by testing of model biological samples.

All the samples (with the concentrations 1х106; 1х105 and 1х104 GE/ml) were prepared

from negative blood plasma with adding the quality control sample (QCS Positive Control

HHV7).

The trueness was determined by testing the quality control sample (QCS Positive Control

HHV7) with the known concentration of HHV7 DNA, which was determined with the use of

QX100™ Droplet Digital™ PCR system (Bio-Rad Laboratories, Inc., USA).

Table 7 Reproducibility

Micro-organism

Initial concentration value, GE/ml

Number of repeats

Average concentration

value, lg

Standard deviation

(SD)

Coefficient of variation

(CV), %

HHV7 1*106 80 5.80 0.07 1.28 HHV7 1*105 80 4.88 0.07 1.44 HHV7 1*104 80 3.92 0.06 1.49

Table 8

Repeatability

Micro-organism

Initial concentration value, GE/ml

Number of repeats

Average concentration

value, lg

Standard deviation

(SD)

The coefficient of

variation (CV), %

HHV7 1*106 40 5.84 0.05 0.89 HHV7 1*105 40 4.95 0.03 0.69 HHV7 1*104 40 3.98 0.05 1.15


Table 9

Trueness

Micro-organism

Number of repeats

Average value of measurement, lg

Specified value, lg Bias (B), %

HHV7 100 8.26 8.23 0.36

13.4. Diagnostic characteristics

Table 10

The results of testing AmpliSens® HHV7-screen/monitor-FRT PCR kit in comparison with the reference assay

Samples type The results of application of

AmpliSens® HHV7-screen/monitor-FRT PCR kit

Results of using the

reference assay3

Positive Negative

Blood plasma 200 samples were tested Positive 100 0

Negative 0 100

Whole blood 200 samples were tested Positive 100 0

Negative 0 100

Saliva 200 samples were tested Positive 100 0

Negative 0 100

The diagnostic sensitivity was estimated by testing 100 model samples of each type of the

biological material (blood plasma, whole blood, saliva), containing dilutions of the quality

control sample of HHV7 DNA. The presence of HHV7 DNA at initial dilutions of the quality

control sample was testified by Sanger sequencing method. The diagnostic specificity was

estimated by testing 100 model samples of each type of the biological material (blood

plasma, whole blood, saliva), obtained from intact donors.

Table 11

Diagnostic characteristics of AmpliSens® HHV7-screen/monitor-FRT PCR kit

Samples type Diagnostic sensitivity4, % Diagnostic specificity 5, %

Blood plasma 100 100

Whole blood 100 100

Saliva 100 100

14. REFERENCES

1. Guidelines to the AmpliSens® HHV7-screen/monitor-FRT PCR kit using the PCR

instruments with real-time hybridization-fluorescence detection developed by Federal

Budget Institute of Science “Central Research Institute for Epidemiology”.

3 Sanger sequencing method were used as reference assay.

4) Relative sensitivity in comparison with applied reference assay.

5) Relative specificity in comparison with applied reference assay.


15. QUALITY CONTROL

In compliance with Federal Budget Institute of Science “Central Research Institute for

Epidemiology” ISO 13485-Certified Quality Management System, each lot of the

AmpliSens® HHV7-screen/monitor-FRT PCR kit has been tested against predetermined

specifications to ensure consistent product quality.

16. KEY TO SYMBOLS USED

Catalogue number

Caution

Batch code

Sufficient for

In vitro diagnostic medical device

Expiration Date

Version

Consult instructions for use

Temperature limitation

Keep away from sunlight

Manufacturer NCA Negative control of amplification

Date of manufacture C– Negative control of extraction

PCE Positive control of extraction

C1, C2 DNA-calibrators

IC Internal control

Documents

PCR diagnostics.eu - HHV7-screen-monitor-FRT ME …...PRINCIPLE OF PCR DETECTION The principle of testing is based on the DNA extraction from test samples together with the exogenous